The agnoprotein is a late SV40 gene encoding a 61 amino acid polypeptide of unknown function. Mutant C1-5, engineered by a site-directed mutagenesis, is a 2-base insertion at the unique HpaII site located within the agnogene. Analysis of plaques formed by transfection of DNA from mutant C1-5 gives evidence of A second mutation. These revertants which occur at high frequency result from base alterations at or near the site of the original mutation and appear to acquire a functional agnoprotein.